Tobacco cultivar ‘AOB 175’

ABSTRACT

The present invention relates to a novel tobacco cultivar designated AOB 175, which has low to intermediate nicotine content. The invention provides seeds of the cultivar AOB 175, plants and parts thereof of the cultivar AOB 175, a tissue culture derived from the cultivar AOB 175, hybrids produced from cultivar AOB 175 and lines derived from cultivar AOB 175, as well as genetically modified forms of the foregoing plants and tissue culture. Also provided are methods of producing cultivar AOB 175 plants, cultivar AOB 175 hybrid plants, and tobacco lines derived from cultivar AOB 175. In addition, products produced from the plants of the present invention are provided.

FIELD OF THE INVENTION

The present invention relates to tobacco breeding, in particular, to anew tobacco cultivar designated AOB 175 having low to intermediatenicotine content.

BACKGROUND OF THE INVENTION

Tobacco (Nicotiana tabacum L.) is an important commercial crop in theUnited States as well as in other countries. The production of tobaccowith decreased levels of nicotine is of interest. Various processes havebeen designed for the removal of nicotine from tobacco. However, most ofthese processes remove other ingredients from tobacco in addition tonicotine, thereby adversely affecting the tobacco.

There are numerous stages in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The aim is to combine in a single variety an improvedcombination of desirable traits from the parental germplasm. Theseimportant traits may include higher yield, resistance to diseases andinsects, better stems and roots, tolerance to drought and heat, improvednutritional quality, and better agronomic characteristics.

Choice of breeding methods depends on the mode of plant reproduction,the heritability of the trait(s) being improved, and the type ofcultivar used commercially (e.g., F₁ hybrid cultivar, pure linecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location may be effective,whereas for traits with low heritability, selection can be based on meanvalues obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences the choice of breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program generally includes a periodic, objectiveevaluation of the efficiency of the breeding procedure. Evaluationcriteria vary depending on the goals and objectives, but should includegain from selection per year based on comparisons to an appropriatestandard, overall value of the advanced breeding lines, and number ofsuccessful cultivars produced per unit of input (e.g., per year, perdollar expended, etc.).

The development of new tobacco hybrids involves the development andselection of tobacco breeding lines, the crossing of these breedinglines and selection of superior hybrid crosses. Hybrid combinations areidentified and developed on the basis of certain single gene traits suchas leaf size or color, flower color, disease resistance or herbicideresistance, and the like, which are expressed in a hybrid. Additionaldata, such as yield and quality traits, on parental lines as well as thephenotype of the hybrid influence the breeder's decision to continuewith the specific hybrid cross.

Pedigree breeding and recurrent selection breeding methods are used todevelop true breeding cultivars from breeding populations. Breedingprograms combine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing or alternatively, by creating doubled-haploids, andselection of desired phenotypes. The new cultivars are evaluated todetermine which have commercial potential.

Pedigree breeding is commonly used for the improvement ofself-pollinating crops and parental lines for hybrids. Two parents whichpossess favorable, complementary traits are crossed to produce an F₁. AnF₂ population is produced by selfing one or several F₁ plants. Selectionof the best individuals may begin in the F₂ population; then, beginningin the F₃, the best individuals in the families are selected. Replicatedtesting of families can begin in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines aretested for potential release as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂, individuals. The number of plants in a population maydecline in each generation due to failure of some seeds to germinate orsome plants to produce at least one seed. As a result, not all of the F₂plants originally sampled in the population will be represented by aprogeny when generation advance is completed.

In a multiple-seed procedure, tobacco breeders harvest seeds from one ormore flowers from each plant in a population and pool them to form abulk. Part of the bulk is used to plant the next generation and part isput in reserve. The procedure has been referred to as modifiedsingle-seed descent technique.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, the breeder should consider whether thereis a demand for a new cultivar that is compatible with industrystandards or which creates a new market. The introduction of a newcultivar will incur additional costs to the seed producer, the grower,the processor and the consumer, for special advertising and marketing,altered seed and commercial production practices, and new productutilization. The testing preceding release of a new cultivar should takeinto consideration research and development costs as well as technicalsuperiority of the final cultivar. For seed-propagated cultivars, itmust be feasible to produce seed easily and economically.

Methods of tobacco breeding are discussed in detail in Wernsman, E. A.,and Rufty, R. C. 1987. Chapter Seventeen. Tobacco. Pages 669-698 In:Cultivar Development. Crop Species. W. H. Fehr (ed.), MacMillanPublishing Go., Inc., New York, N.Y. 761 pp.

SUMMARY OF THE INVENTION

The present invention relates to a new and distinctive tobacco cultivardesignated AOB 175 having desirable agronomic and smokingcharacteristics in combination with low to intermediate nicotinecontent.

The invention further provides seeds of the cultivar AOB 175, plants ofthe cultivar AOB 175 and parts thereof, for example, leaves, pollen,embryos, cotyledons, hypocotyls, roots, root tips, anthers, flowers,ovules, shoots, stems, stalks, pith and capsules, tissue culturecomprising tissue, callus, cells or protoplasts of the cultivar AOB 175,hybrids having a cultivar AOB 175 parent or ancestor, and AOB 175derived tobacco plants, as well as genetically modified (e.g., byconventional breeding or genetic engineering techniques) forms of theforegoing plants and tissue culture.

The present invention further provides methods of producing a tobaccoplant by crossing the AOB 175 cultivar with itself or a differenttobacco line. The invention further relates to methods for producingother tobacco cultivars or breeding lines derived from the cultivar AOB175 by crossing the AOB 175 cultivar with a second tobacco plant andgrowing the progeny seed to yield an AOB 175-derived tobacco plant. Anadditional embodiment of the invention provides a method for a tobaccoplant that contains in its genetic material one or more transgenes,comprising crossing an AOB 175 cultivar containing one or moretransgenes with either a second plant of another tobacco line, or anon-transformed AOB 175 tobacco plant, wherein progeny are produced, sothat the genetic material of the progeny that result from the crosscomprise the transgene(s) optionally operably linked to one or moreregulatory elements.

Another aspect of the invention provides a method for developing atobacco plant in a tobacco plant breeding program using plant breedingtechniques, which includes employing an AOB 175 tobacco plant or a partthereof, or an AOB 175-derived tobacco plant, or a part thereof, as asource of plant breeding material, wherein the plant breeding techniquesare selected from the group consisting of recurrent selection,backcrossing, pedigree breeding, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selection,double haploid breeding, single seed descent, multiple seed descent, andtransformation.

A further aspect of the present invention provides products comprisingtobacco wherein the tobacco further comprises tobacco from the plants ofthe present invention, and parts thereof.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter withreference to the accompanying drawings and specification, in whichpreferred embodiments of the invention are shown. This invention may,however, be embodied in different forms and should not be construed aslimited to the embodiments set forth herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention.

All publications, patent applications, patents and other referencescited herein are incorporated by reference in their entireties for theteachings relevant to the sentence and/or paragraph in which thereference is presented.

As used in the description of the invention and the appended claims, thesingular forms “a”, “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

The term “about,” as used herein when referring to a measurable valuesuch as an amount of a compound (e.g., an amount of nicotine) and thelike, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%,or even ±0.1% of the specified amount.

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim, “and those that donot materially affect the basic and novel characteristic(s)” of theclaimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q.461, 463 (CCPA 1976) (emphasis in the original); see also MPEP §2111.03.Thus, the term “consisting essentially of” when used in a claim of thisinvention is not intended to be interpreted to be equivalent to“comprising.”

As used herein, the term “plant” includes plant cells, plant protoplastsand plant tissue (e.g., in culture; tissue culture) from which tobaccoplants can be regenerated, plant calli, plant clumps, and plant cellsthat are intact in plants or parts of plants, such as leaves, pollen,embryos, cotyledon, hypocotyl, roots, root tips, anthers, flowers and apart thereof, ovules, shoots, stems, stalks, pith, capsules, and thelike.

As used herein, the term “tissue culture” encompasses cultures oftobacco tissue, cells, protoplasts and callus. Methods of culturingtobacco tissue, cells, protoplasts and callus, as well as methods ofregenerating plants from tobacco tissue cultures are described inWernsman, E. A., and Rufty, R. C. 1987. Chapter Seventeen. Tobacco.Pages 669-698 In: Cultivar Development. Crop Species. W. H. Fehr (ed.),MacMillan Publishing Go., Inc., New York, N.Y. 761 pp.

As used herein, the term “resistance” and the term “tolerance” refer tothe ability of a plant to withstand exposure to an insect, a disease orpathogen, an herbicide or other agent or condition (abiotic or biotic).A resistant or tolerant plant variety will have a level of resistance ortolerance, respectively, that is higher than a comparable wild-typevariety.

Description of the Variety.

A breeder uses various methods to help determine which plants should beselected from segregating populations and ultimately which inbred lineswill be used to develop hybrids for commercialization. In addition toknowledge of the germplasm and plant genetics, a part of the selectionprocess is dependent on experimental design coupled with the use ofstatistical analysis. Experimental design and statistical analysis areused to help determine which plants, which family of plants, and finallywhich inbred lines and hybrid combinations are significantly better ordifferent for one or more traits of interest.

In the case of the present tobacco variety, AOB 175, selection for eachgeneration was initially made based on field observations of variousphenotypic characteristics such as degree of maturity, number of leavesper plant, leaf insertion angle, leaf size (width and length), internodedistance, and lamina-midrib ratio. The leaves of the selected plantswere harvested, cured and then analyzed for nicotine content. The plantsfinally selected for each generation were those having reduced nicotinecontent as described below.

AOB 175 was the result of an initial cross between the tobacco varietiesLI BY 21 and TN 90, which was carried out in Vera Cruz/Rio Grande do Sul(Latitude: 29°42′53″S; Longitude: 52°30′20″W), Brazil during the cropseason of 1997/1998. The F₁ seeds were sown in the greenhouse during thewinter season of 1998 and the F₂ seeds were produced throughself-pollination of the F₁ plants.

In the 1999/2000 crop season, the F₂ seeds were sown and 90 plants weretransplanted into the field in Vera Cruz/Rio Grande do Sul, Brazil. Ofthese 90 plants, 50 were selected based on field observations asdescribed above. The leaves from these 50 plants were harvested, curedand then analyzed for their nicotine content. Those plants with nicotinelevels falling between the population average and the population averageminus one standard deviation were selected to give rise to the F₃generation. Accordingly, based on the results of the nicotine analysis,six F₂ plants were selected to give rise to six separate F₃ families(designated as 00/LABS-25; 00/LABS-27; 00/LABS-28; 00/LABS-32;00/LABS-33; and 00/LABS-34). For the variety AOB 175, the 00/LABS-32family formed the F₃ generation.

In the 2000/2001 crop season, the 00/LABS-32 F₃ seeds were sown and 70plants were transplanted into a field in Vera Cruz/Rio Grande do Sul,Brazil. Fifty plants were selected from the 70 F₃ plants based onphenotypic characteristics, as described above, and their leaves wereanalyzed for nicotine content. Those plants with nicotine levels fallingbetween the population average and the population average minus onestandard deviation were selected to give rise to the F₄ generation.Accordingly, based on the results of the nicotine analysis, five of thefifty F₃ plants were selected and their seeds were bulked and used toform two separate F₄ populations (designated 01/LABS-19 and 01/LABS-20).

In the 2001/2002 crop season, the 01/LABS-20 F₄ seeds were sown and 50F₄ plants were transplanted into a field in Vera Cruz/Rio Grande do Sul,Brazil. Twenty-five plants were then selected based on phenotypiccharacteristics, as described above, and their leaves were analyzed fornicotine content. Those plants with nicotine levels falling between thepopulation average plus one standard deviation and the populationaverage minus one standard deviation were selected to give rise to theF₅ generation. Thus, based on the nicotine analysis results, nine out ofthe twenty-five F₄ plants were selected and their seeds bulked, givingrise to the F₅ generation (02/LABS-17).

During the 2002/2003 crop season, the 02/LABS-17 seeds were sown and 60F₅ plants were transplanted into the field at Palmitos/Santa Catarina,Brazil (Latitude: 27°04′03″S; Longitude: 53°09′40″W). Of the 60 F₅plants, 20 were selected based on phenotypic characteristics observed inthe field, as described above and their leaves analyzed for nicotinecontent. Those plants with nicotine levels falling between thepopulation average plus one-half the standard deviation and thepopulation average minus one-half the standard deviation were selectedto give rise to the F₆ generation. Accordingly, based on the results ofthe nicotine analysis, thirteen of the F₅ plants were selected and theirseeds bulked, giving rise to the F₆ generation (designated 03/LABS-175).

In the 2003/2004 crop season, the 03/LABS-175 F₆ seeds were sown and 60plants were transplanted into the field at Palmitos/Santa Catarina,Brazil. Out of the 60 plants, 20 were selected as described previouslyand analyzed for their nicotine content. Those plants with nicotinelevels falling between the population average plus one-half the standarddeviation and the population average minus one-half the standarddeviation were selected to give rise to the F₇ generation. Accordingly,seven plants out of the twenty F₆ plants were selected and their seedsbulked, giving rise to the F₇ generation (designated LABS 175 or AOB175).

During the 2004/2005 crop season, the AOB 175 F₇ seeds were sown and 144F₇ plants were transplanted into the field at Palmitos/Santa Catarina,Brazil. Of the 144 plants, twenty were selected based on phenotypiccharacteristics, as described previously, and their leaves analyzed fornicotine content. Similar to the previous two generations, those plantswith nicotine levels falling between the population average plusone-half the standard deviation and the population average minusone-half the standard deviation were selected to give rise to the nextgeneration (F₈). Thus, based on the results of the nicotine analysis,seven F₇ plants were selected and their seeds were bulked, giving riseto the F₈ generation and to the foundation seed of AOB 175.

The variety AOB 175, having been observed for eight generations, isconsidered uniform and stable. The variety AOB 175 shows no variantplants other than what would normally be expected due to environmentalconditions.

Table 1 provides morphological data and other characteristics of thevariety AOB 175.

TABLE 1 AOB 175 Variety Description Information. Class 3 (Burley)Maturity Class 3 (late) Days to Maturity 83 Height Class 3 (tall) PlantHeight (cm) Topped Normal 163 Not Topped (height to crowfoot) 206 LeafLength (cm)  5th leaf 64 10th leaf 66 15th leaf 63.4 Leaf Length Class(10^(th) leaf or center of plant) 2 (medium) Leaf Width (cm)  5th leaf31 10th leaf 30.6 15th leaf 28 Leaf Width Class (10^(th) leaf or centerof plant) 3 (medium) Leaf Angle (degrees)  5th leaf 77 10th leaf 72 15thleaf 62 Leaf Angle Class (10^(th) leaf or center of plant) 4 (drooping)Leaf Yield (Kg/ha) 2,950 Leaf Number per plant (not including 2 bedleaves) Topped Normal 21.6 Not Topped (number of leaves or nodes to 25.8crowfoot from first harvestable leaf) Internode Length Class 1 (short)Internode Length (mm) 46 Stalk Diameter Class 1 (small) Leaf CarriageNot Arched Tip Shape Acuminate Leaf Margin Wavy Leaf Color GreenVenation Pattern Square Leaf Margin Curving Not Recurved Leaf ShapeBroadcast at middle of leaf Leaf Surface Puckered Flowers Color PinkHead Habit Intermediate Plant Form Pyramidal Ground Suckers (per plant)0.2 Disease Bacterial Wilt Susceptible Potato Virus Y SusceptibleTobacco Vein Mottling Virus Susceptible Tobacco Mosaic Virus HighResistance Tobacco Etch Virus Susceptible Leaf Constituents % Nicotine3.66 % Nor Nicotine 0.08 % Total Nitrogen 3.49

The classes for specific characteristics that are set forth in Table 1are those defined by the United States Plant Variety Protection Office(See, Exhibit C for tobacco).

Further characteristics of AOB 175 are provided in Tables 2-4, whichcompare yield, grade index and nicotine content of the new variety witheach of its parents, the tobacco varieties LI BY 21 and TN 90 overseveral different growing seasons. In each case the asterix (*)indicates no significant differences were observed between the averageswith the same letter in the same column (by DUNCAN test at 5%probability).

Table 2 presents data from the 2004/05 crop season in which experimentswere conducted at two locations: Pinhalzinho/Santa Catarina (Latitude:26°50′53″S; Longitude: 52°59′31″W) and Vila Maria/Rio Grande do Sul(Latitude: 28°32′05″S; Longitude: 52°09′13″W). The experiments wereconducted following a randomized design with three repetitions and 48plants to each plot. The plants were spaced 45 cm apart with 115 cmbetween lines (19,523 plants/ha). The total nitrogen used was 234 and247 kg/ha, respectively. Harvesting occurred at 45 and 42 days aftertopping, respectively.

TABLE 2 Mean yield, grade index and nicotine of check cultivars an AOB175 grown at the Pinhalzinho/SC and Vila Maria/RS, Brazil, during the2004/05 crop season. Variety Yield (Kg/ha) Grade Index Nicotine (%)Pinhalzinho/Santa Catarina TN 90 3,311 a* 59.5 a 5.26 a LI BY 21 2,949 a49.3 a 2.60 b AOB 175 3,183 a 48.0 a 2.64 b Vila Maria/Rio Grande do SulTN 90 3,164 a 68.3 a 5.77 a LI BY 21 2,501 b 51.6 b 3.08 b AOB 175 2,692b 65.2 a 3.31 b AVERAGE CROP 2004/05 TN 90 3,238 a 63.9 a 5.52 a LI BY21 2,725 b 50.5 b 2.84 b AOB 175 2,938 b 56.6 ab 2.98 b

Table 3 presents data from the 2005/2006 crop season. Experiments wereconducted at three different locations in Brazil: Palmitos/SantaCatarina, Pinhalzinho/Santa Catarina and Anta Gorda/Rio Grande do Sul(Latitude: 28°53′41″S; Longitude: 52°02′09″W). The experiments followedthe same design as set forth above for the 2004/2005 growing season,except that the plot size changed from 44 to 30 plants/plot. The totalnitrogen used was 224, 212 and 248 kg/ha, respectively. Harvesting tookplace at 48, 41 and 53 days after topping, respectively.

TABLE 3 Mean yield, grade index and nicotine of check cultivars and AOB175 grown at Palmitos/SC, Pinhalzinho/SC and Anta Gorda/RS, Brazil,during the 2005/06 crop season. Variety Yield (Kg/ha) Grade IndexNicotine (%) Palmitos/Santa Catarina TN 90 3,588 a* 70.3 a 5.79 a LI BY21 2,766 a 57.9 a 3.29 b AOB 175 3,269 a 53.0 a 3.64 b Pinhalzinho/SantaCatarina TN 90 2,813 a 71.9 a 5.62 a LI BY 21 2,661 a 62.4 a 2.14 b AOB175 2,865 a 64.4 a 2.96 b Anta Gorda/Rio Grande do Sul TN 90 3,049 a79.8 a 5.74 a LI BY 21 2,546 a 77.4 a 2.86 b AOB 175 2,884 a 73.1 a 3.82ab AVERAGE CROP 2005/06 TN 90 3,150 a 74.0 a 5.72 a LI BY 21 2,658 a65.9 a 2.76 b AOB 175 3,006 a 63.5 a 3.48 b

During the 2006/2007 crop season, an experiment was conducted VeraCruz/Rio Grande do Sul, Brazil (Latitude: 29°42′53″S; Longitude:52°30′20″W) to evaluate nicotine and total of nitrogen of the newvariety, AOB 175, as compared to the parental varieties, TN 90 and LI BY21. These data are shown in Table 4.

TABLE 4 Mean total alkaloids and total nitrogen of check cultivars andAOB 175, grown in the Research Center of Alliance One, Vera Cruz/RS,Brazil, during the 2006/07 crop season. Variety Nicotine (%) TotalNitrogen (%) TN 90 5.55 a* 4.23 a LI BY 21 3.31 b 3.57 a AOB 175 3.22 b3.49 a

As shown in Tables 2, 3 and 4, the nicotine levels of the new varietyAOB 175 were determined to be significantly lower than that for TN 90but similar to LI BY 21. In contrast, the total nitrogen content of allthree varieties was similar (Table 4). The average yield for AOB 175over both crop seasons and all growing sites was generally intermediateto, but not statistically different from either parent, TN 90 or LI BY21 (Tables 2 and 3). In the 2004/2005 crop season, the average gradeindex for AOB 175 was lower than TN 90 and similar to LI BY 21 (Table2), but during the 2005/2006 crop season, yield and grade index weresimilar for all three varieties (Table 3).

Thus, the new tobacco variety AOB 175 shows different phenotypiccharacteristics when compared to its progenitors, TN 90 and LI BY 21. Ofsignificance, and as discussed above, AOB 175 produces less nicotinethan the parent, TN 90. Specifically, AOB 175 produces about 40 to 45%less nicotine compared with TN 90. In addition, AOB 175 can bedistinguished from TN 90 by its susceptibility to Potato Virus Y (PVY),whereas TN 90 is resistant.

Further, when compared with LI BY 21, another important distinctivecharacteristic of AOB 175 is the rate of maturation with AOB 175maturing about 15 days later than LI BY 21. Additionally, the untoppedplant height of AOB 175 is approximately 25 cm greater than LI BY 21.The leaves of AOB 175 are about the same width as those of LI BY 21, butaverage about 5 cm longer. Also, AOB 175 has a more consistent leaf sizeand shape than LI BY 21. The leaf insertion angle for AOB 175 is similarto LI BY 21. In untopped plants, AOB 175 averages five leaves more thanLI BY 21. Additionally, the leaf surface is of AOB 175 is puckered(Exhibit C—Class 2) while that of LI BY 21 is smooth (Exhibit C—Class1). The AOB 175 leaf margin is wavy (Exhibit C—Class 1) whereas LI BY21, has a leaf margin that is not wavy (Exhibit C—Class 2). AOB 175 isoverall a more compact plant than LIBY 21, thus allowing for less damageduring harvest. In addition, AOB 175 tolerates colder climates betterthan LI BY 21.

When comparing AOB 175 with its sister lines, AOB 174 and AOB 176, AOB175 is the latest maturing, being about 5 days later than AOB 174 and 10days later than AOB 176. In untopped plants, AOB 175 is 5 cm taller thanAOB 174 and 20 cm taller than AOB 176. The leaf size of AOB 175 isintermediate to its two sister lines, being about 2 cm broader and 5 cmlonger than AOB 174 and 2 cm narrower and 2 cm shorter than AOB 176. Theleaf insertion angle for AOB 175 is similar to AOB 174, but is largerthan AOB 176. Compared with its sister lines, AOB 175 averages five moreleaves in untopped plants than AOB 176 and about three more leaves thanAOB 174. The leaf surface of AOB 175 is puckered (Exhibit C—Class 2)while the leaf surface of AOB 174 is smooth (Exhibit C—Class 1). Inaddition, AOB 175 has a leaf margin that is wavy (Exhibit C—Class 1),similar to AOB 174, but different from AOB 176, which is not wavy(Exhibit C—Class 2).

Thus, AOB 175 is a new and distinct tobacco variety, differing from bothparental varieties, TN 90 and LI BY 21, and from its sister lines, AOB174 and AOB 176. AOB 175, with similar yield and quality to TN 90,provides the additional advantage over TN 90 of producing less nicotineand thus, is a good alternative for producing tobacco with less nicotinecontent. In addition, compared with its sister lines, AOB 174 and AOB176, AOB 175 is appropriate for growing in a normal season mainly due toits intermediate maturation rate and leaf number.

Table 5 provides a comparison of various characteristics of the newvariety AOB 175 with those of the known tobacco varieties Kentucky 14(KY 14) and Burley 21 (BY 21).

TABLE 5 Comparison of AOB 175 with the tobacco varieties BY 21 and KY14. Variety Characteristic AOB 175 BY 21 KY 14 Maturity-Flowering 83 6873 Plant Height (cm) Topped 163 128 144 Not Topped 206 179 184 LeafLength  5th leaf 64 62.6 62.2 10th leaf 66 61.6 67.8 15th leaf 63.4 56.865.8 Leaf Width (cm)  5th leaf 31 33 34.8 10th leaf 30.6 29.2 34 15thleaf 28 26 27.2 Leaf Angle (degrees)  5th leaf 77 81 48 10th leaf 72 6547 15th leaf 62 60 56 Leaf Number per plant Topped Normal 21.6 20.4 21.8Not Topped 25.8 21.6 24 Internode Length (mm) 46 48 54 % Nicotine 3.665.12 % Nor Nicotine 0.08 0.09 % Total Nitrogen 3.49 3.92

The goal of this TN 90 and LI BY 21 cross was to develop a new varietywith low to intermediate nicotine content and with agronomic and smokingcharacteristics desirable to farmers and to the tobacco industry. Thus,the AOB 175 variety shows TMV resistance, high yield and quality withlower nicotine levels than that observed for the parental variety, TN90.

Accordingly, one aspect of the present invention is a tobacco seeddesignated AOB 175. Another aspect of the invention is a tobacco plant,or a part thereof, produced by the seed of the tobacco cultivar AOB 175.A further aspect of the invention is pollen or an ovule of a tobaccoplant produced by the seed of AOB 175. In addition, the presentinvention provides a tobacco plant, or a part thereof, produced by theseed of AOB 175, wherein the tobacco plant further comprises a nucleicacid conferring male sterility.

The present invention additionally provides a tobacco plant, or a partthereof, having all the physiological and morphological characteristicsof tobacco cultivar AOB 175. In other aspects of the invention, thetobacco plant, or a part thereof, having all the physiological andmorphological characteristics of tobacco cultivar AOB 175, furthercomprises a nucleic acid conferring male sterility.

Still further, the invention provides a tissue culture of regenerablecells of the plant, or part thereof, of the present invention, whichculture regenerates tobacco plants capable of expressing all themorphological and physiological characteristics of tobacco cultivar AOB175. The regenerable cells of the invention include but are not limitedto cells from leaves, pollen, embryos, cotyledons, hypocotyls, roots,root tips, anthers, flowers and a part thereof, ovules, shoots, stems,stalks, pith and capsules or callus or protoplasts derived therefrom.Thus, another aspect of this invention is to provide cells, which upongrowth and differentiation produce tobacco plants having thephysiological and morphological characteristics of tobacco cultivar AOB175. In some embodiments, cells of cultivar AOB 175 are transformedgenetically, for example with one or more nucleic acids described below,and transgenic plants of tobacco cultivar AOB 175 are regeneratedtherefrom.

AOB 175 has shown uniformity and stability within the limits ofenvironmental influence for all the traits as described in the VarietyDescription Information (Table 1). A sufficient number of generationshave been observed with careful attention paid to uniformity of planttype to ensure the homozygosity and phenotypic stability necessary foruse in commercial hybrid seed production. No variant traits have beenobserved or are expected in AOB 175.

OTHER EMBODIMENTS OF THE INVENTION

The present invention also encompasses hybrid plants produced fromtobacco cultivar AOB 175, tobacco plants derived from AOB 175, and AOB175 plants comprising a nucleic acid that has been introduced therein bytraditional breeding or genetic engineering techniques, and seeds, plantparts, and tissue cultures of the foregoing plants, as well as methodsof producing the plants of the invention.

Accordingly, methods for crossing the tobacco plants of the presentinvention are provided. Such methods may comprise crossing the plant ofthe present invention, AOB 175, with itself or a second tobacco plant.The present invention further encompasses a method for producing hybridtobacco seed, the method comprising crossing two tobacco plants andharvesting the resultant hybrid tobacco seed, wherein at least onetobacco plant is the tobacco plant of the present invention, AOB 175. Inanother embodiment, a method for producing a first generation (F₁)hybrid tobacco seed is provided comprising crossing the plant of thepresent invention with a different tobacco plant and harvesting theresultant first generation (F₁) hybrid tobacco seed. Further provided bythe present invention are plants produced by these methods.

Additionally provided herein, is a method for producing an AOB175-derived tobacco plant comprising: (a) crossing tobacco cultivar AOB175 with a second tobacco plant to yield progeny tobacco seed; (b)growing said progeny tobacco seed, under plant growth conditions, toyield said AOB 175-derived tobacco plant. The method may still furthercomprise: a) crossing said AOB 175-derived tobacco plant with itself oranother tobacco plant to yield additional AOB 175-derived progenytobacco seed; (b) growing said progeny tobacco seed of step (a) underplant growth conditions, to yield additional AOB 175-derived tobaccoplants; and (c) repeating the crossing and growing steps of (a) and (b)multiple times to generate further AOB 175-derived tobacco plants. Insome embodiments, the crossing and growing steps of (a) and (b) in step(c) are repeated from 0 to 2 times, from 0 to 3 times, from 0 to 4times, 0 to 5 times, from 0 to 6 times, from 0 to 7 times, from 0 to 8times, from 0 to 9 times or from 0 to 10 times, in order to generatefurther AOB 175-derived tobacco plants. In other embodiments, thecrossing and growing steps of (a) and (b) in step (c) are repeated from0 to n times in order to generate further AOB 175-derived tobaccoplants. The invention further provides plants produced by these methods.Accordingly, the invention encompasses progeny plants and parts thereofwith at least one ancestor that is hybrid tobacco plant AOB 175 and morespecifically where the pedigree of this progeny includes 1, 2, 3, 4, 5,6, and/or 7 cross pollinations to a tobacco plant AOB 175 or a plantthat has AOB 175 as a progenitor.

Other embodiments of the present invention include a method forproducing a tobacco plant that contains in its genetic material one ormore transgenes, comprising crossing the tobacco plant of the presentinvention with either a second plant of another tobacco line, or anon-transformed tobacco plant of the present invention, wherein progenyare produced, so that the genetic material of the progeny that resultfrom the cross comprises the transgene(s) operably linked to one or moreregulatory elements. In one aspect of the invention, the one or moretransgene includes but is not limited to a nucleic acid conferringherbicide resistance, insect resistance, disease resistance and/or malesterility. Further provided by the present invention are plants producedby this method.

Further provided by the present invention is a method for developing atobacco plant in a tobacco plant breeding program using plant breedingtechniques, which include employing a tobacco plant of the presentinvention, or a part thereof, as the source of plant breeding material.Plant breeding techniques that can be used in the method include, butare not limited to, recurrent selection, backcrossing, pedigreebreeding, restriction fragment length polymorphism enhanced selection,genetic marker enhanced selection, double haploid breeding, single seeddescent, multiple seed descent, and/or transformation. Further providedherein are plants produced by this method.

Accordingly, any methods using the cultivar AOB 175 are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using cultivar AOB 175 asa parent are within the scope of this invention including plants derivedfrom the cultivar AOB 175. Advantageously, AOB 175 cultures used incrosses with other tobacco cultivars can be used to produce a firstgeneration (F1) tobacco hybrid seed and plants with superiorcharacteristics.

I. MALE STERILE PLANTS

Tobacco can be bred by both self-pollination and cross-pollinationtechniques. Individual tobacco flowers have both male and femalereproductive organs, and tobacco is naturally self-pollinating. It isknown in the art that it is often advantageous to create malesterile/female fertile plants so that self-pollination can becontrolled.

Male sterile tobacco plants may be produced by any method known in theart. Methods of producing male sterile tobacco are described inWernsman, E. A., and Rufty, R. C. 1987. Chapter Seventeen. Tobacco.Pages 669-698 In: Cultivar Development. Crop Species. W. H. Fehr (ed.),MacMillan Publishing Go., Inc., New York, N.Y. 761 pp.

A reliable method of controlling male fertility in plants offers theopportunity for improved plant breeding. This is especially true fordevelopment of tobacco hybrids, which typically relies upon some sort ofmale sterility system. There are several options for controlling malefertility available to breeders, such as: manual or mechanicalemasculation, cytoplasmic male sterility, genetic male sterility,gametocides and the like. In one approach, alternate strips of twotobacco lines are planted in a field, and the male portions of flowersare removed from one of the lines (female). Providing that there issufficient isolation from sources of foreign tobacco pollen, theemasculated plant will be fertilized only from the other line (male),and the resulting seed is therefore hybrid and will form hybrid plants.

The laborious, and occasionally unreliable, mechanical emasculationprocess can be avoided by using cytoplasmic male-sterile (CMS) lines.Plants of a CMS line are male sterile as a result of factors resultingfrom the cytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent intobacco plants, since only the female provides cytoplasm to thefertilized seed. CMS plants are fertilized with pollen from another linethat is not male-sterile. Pollen from the second line may or may notcontribute genes that make the hybrid plants male-fertile.

Alternative approaches of conferring genetic male sterility are alsosuitable, such as multiple mutant nucleic acids at separate locationswithin the genome that confer male sterility and chromosomaltranslocations.

Still further methods of conferring genetic male sterility use a varietyof approaches such as delivering into the plant a nucleic acid encodinga cytotoxic substance associated with a male tissue specific promoter oran antisense system in which a nucleic acid critical to male fertilityis identified and an antisense to that nucleic acid is inserted in theplant.

Another system useful in controlling male fertility makes use ofgametocides. Gametocides do not involve a genetic system, but rather atopical application of chemicals. These chemicals affect cells that arecritical to male fertility. The application of these chemicals affectsfertility in the plants only for the growing season in which thegametocide is applied (see U.S. Pat. No. 4,936,904). Application of thegametocide, timing of the application and genotype specificity oftenlimit the usefulness of the approach.

II. HYBRID PRODUCTION

The use of male sterile lines is one factor in the production of tobaccohybrids. The development of tobacco hybrids involves, in general, thedevelopment of completely homozygous lines, the crossing of these lines,and the evaluation of the crosses. In the case of tobacco, a completelyhomozygous line may be an inbred or a doubled-haploid line.

Pedigree breeding and recurrent selection breeding methods are typicallyused to develop inbred lines from breeding populations. Breedingprograms combine the genetic backgrounds from two or more inbred linesor various other germplasm sources into breeding pools from which newinbred lines are developed by selfing and selection of desiredphenotypes. The new inbreds are crossed with other inbred lines ordoubled-haploid lines, and the hybrids from these crosses are evaluatedto determine which have commercial potential.

Pedigree breeding starts with the crossing of two genotypes, each ofwhich may have one or more desirable characteristics that is lacking inthe other or which complements the other. If the two original parents donot provide all the desired characteristics, other sources can beincluded in the breeding population. In the pedigree method, superiorplants are selfed and selected in successive generations. In thesucceeding generations, the heterozygous condition gives way tohomogeneous lines as a result of self-pollination and selection.Typically in the pedigree method of breeding, five or more generationsof selfing and selection is practiced. Thus, multiple crossings andgrowing steps may be carried out in order to generate a desired hybrid.

A single cross tobacco hybrid results from the cross of two tobaccolines (e.g., inbred or doubled-haploid lines), each of the parentshaving a genotype that complements the genotype of the other. The hybridprogeny of the first generation is designated F₁. Preferred F1 hybridsmay be more vigorous than either parent in a cross between inbredparents. This hybrid vigor, or heterosis, can be manifested in manypolygenic traits, including increased vegetative growth and increasedyield.

In general, the development of a tobacco hybrid involves three steps:(1) the selection of plants from various germplasm pools for initialbreeding crosses; (2) the selfing of the selected plants from thebreeding crosses for several generations to produce a series of inbredlines, which, although different from each other, breed true and arehighly uniform; and (3) crossing the selected inbred lines withdifferent inbred lines to produce the hybrid progeny (F₁). A consequenceof the homozygosity and homogeneity of the inbred lines is that thehybrid between a defined pair of inbreds/doubled-haploids will always bethe same. Once the parents that give a superior hybrid have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the parents is maintained.

A single cross hybrid is produced when two lines are crossed to producethe F₁ progeny. A double cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F₁ hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor exhibited by F₁ hybrids isgenerally lost in the next generation (F₂). Consequently, seed fromhybrids is not typically used for planting stock.

As described above, hybrid seed production regimes generally use malesterile/female fertile parent plants. Incomplete removal or inactivationof the pollen provides the potential for self pollination. Thisinadvertently self pollinated seed may be unintentionally harvested andpackaged with hybrid seed. Once the seed is planted, it is possible toidentify and select these self pollinated plants due to their decreasedvigor. These self-pollinated plants will be genetically equivalent tothe female inbred line used to produce the hybrid. Female selfs areidentified by their less vigorous appearance for vegetative and/orreproductive characteristics as is known in the art.

Identification of these self-pollinated lines can also be accomplishedthrough molecular marker analyses. Through these technologies, thehomozygosity of the self-pollinated line can be verified by analyzingallelic composition at various loci along the genome.

III. EVALUATION OF PLANTS FOR HOMOZYGOSITY AND PHENOTYPIC STABILITY

It is desirable and advantageous for a tobacco cultivar to be highlyhomogeneous, homozygous and phenotypically uniform and stable for use asa commercial cultivar. In the case of inbreds or other lines, there aremany analytical methods available to determine the homozygotic andphenotypic stability of the variety.

The oldest and most traditional method of analysis is the observation ofphenotypic traits. The data are usually collected in field experimentsover the life of the tobacco plants to be examined. Phenotypiccharacteristics most often observed are for traits associated with seedyield, disease resistance, maturity, plant height, internode distance,flower color, leaf color, leaf yield, leaf size, leaf angle,lamina-midrib ratio, and concentration of chemical components such asnicotine, total alkaloids or reducing sugars.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotypes; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), and Simple SequenceRepeats (SSRs) which are also referred to as Microsatellites.

The presence or absence of a marker in the plant genotype may bedetermined by any method known in the art. For example, the markersequence (or its complement) may be used as a hybridization probe, e.g.,for Southern or in situ analysis of genomic DNA. Typically, however, dueto greater ease and sensitivity, an amplification method, such as PCRwill be used to detect the presence or absence of the marker in theplant genotype.

Molecular markers can be used in any method of nucleic acidamplification known in the art. Such methods include but are not limitedto Polymerase Chain Reaction (PCR; described in U.S. Pat. Nos.4,683,195; 4,683,202; 4,800,159; 4,965,188), Strand DisplacementAmplification (SDA; described by G. Walker et al., Proc. Nat. Acad. Sci.USA 89:392 (1992); G. Walker et al., Nucl. Acids Res. 20:1691 (1992);U.S. Pat. No. 5,270,184), thermophilic Strand Displacement Amplification(tSDA; EP 0 684 315 to Frasier et al.), Self-Sustained SequenceReplication (3SR; J. C. Guatelli et al., Proc Natl. Acad. Sci. USA 87:1874-78 (1990)), Nucleic Acid Sequence-Based Amplification (NASBA; U.S.Pat. No. 5,130,238 to Cangene), the Op replicase system (P. Lizardi etal., BioTechnology 6: 1197 (1988)), or transcription based amplification(D. Y. Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173-77 (1989)).

IV. TRANSFER OF TRAITS INTO TOBACCO CULTIVAR AOB 175

Genetic variants of AOB 175 that are naturally-occurring or createdthrough traditional breeding methods using cultivar AOB 175 are alsointended to be within the scope of this invention. In particularembodiments, the invention encompasses plants of cultivar AOB 175 andparts thereof further comprising one or more additional traits, inparticular, specific, single gene transferred traits. Examples of traitsthat may be transferred include, but are not limited to, herbicideresistance, disease resistance (e.g., bacterial fungal or viraldisease), nematode resistance, tolerance to abiotic streses (e.g.,drought, temperature, salinity), yield enhancement, improved nutritionalquality (e.g., oil starch and protein content or quality), alteredchemical composition (e.g., nicotine, secondary alkaloids, totalalkaloids, reducing sugars), improved leaf characteristics (color,shape, size, number, angle), altered reproductive capability (e.g., malesterility) or other agronomically important traits.

Such traits may be introgressed into cultivar AOB 175 from anothertobacco cultivar or may be directly transformed into cultivar AOB 175(discussed below). One or more new traits can be transferred to cultivarAOB 175, or, alternatively, one or more traits of cultivar AOB 175 arealtered or substituted. The introgression of the trait(s) into cultivarAOB 175 may be achieved by any method of plant breeding known in theart, for example, pedigree breeding, backcrossing, doubled-haploidbreeding, and the like (see, Wernsman, E. A., and Rufty, R. C. 1987.Chapter Seventeen. Tobacco. Pages 669-698 In: Cultivar Development. CropSpecies. W. H. Fehr (ed.), MacMillan Publishing Co., Inc., New York,N.Y. 761 pp.).

The laboratory-based techniques described above, in particular RFLP andSSR, can be used in such backcrosses to identify the progenies havingthe highest degree of genetic identity with the recurrent parent. Thispermits one to accelerate the production of tobacco cultivars having atleast 90%, preferably at least 95%, more preferably at least 99% geneticidentity with the recurrent parent, yet more preferably geneticallyidentical to the recurrent parent, and further comprising the trait(s)introgressed from the donor parent. Such determination of geneticidentity can be based on molecular markers used in the laboratory-basedtechniques described above.

The last backcross generation can be selfed to give pure breedingprogeny for the nucleic acid(s) being transferred. The resulting plantsgenerally have essentially all of the morphological and physiologicalcharacteristics of cultivar AOB 175, in addition to the transferredtrait(s) (e.g., one or more single gene traits). The exact backcrossingprotocol will depend on the trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the trait being transferred is a dominant allele, arecessive allele may also be transferred. In this instance, it may benecessary to introduce a test of the progeny to determine if the desiredtrait has been successfully transferred.

Those skilled in the art will appreciate that the tobacco nucleic acidsdescribed below in connection with tobacco plants produced by geneticengineering techniques may also be introduced into cultivar AOB 175 byconventional breeding methods.

V. TRANSFORMATION OF TOBACCO

With the advent of molecular biological techniques that have allowed theisolation and characterization of nucleic acids that encode specificprotein products, scientists in the field of plant biology developed astrong interest in engineering the genome of plants to contain andexpress foreign nucleic acids, or additional, or modified versions ofnative or endogenous nucleic acids (perhaps driven by differentpromoters) in order to alter the traits of a plant in a specific manner.Such foreign, additional and/or modified nucleic acids are referred toherein collectively as “transgenes.” The term “transgene,” as usedherein, is not necessarily intended to indicate that the foreign nucleicacid is from a different plant species. For example, the transgene maybe a particular allele derived from another tobacco line or may be anadditional copy of an endogenous gene. Over the last twenty totwenty-five years several methods for producing transgenic plants havebeen developed. Therefore, in particular embodiments, the presentinvention also encompasses transformed versions of the tobacco cultivarAOB 175.

Plant transformation generally involves the construction of anexpression vector that will function in plant cells. Such a vectorcomprises DNA or RNA comprising a nucleic acid under control of, oroperatively linked to, a regulatory element (for example, a promoter).The expression vector may contain one or more such operably linkednucleic acid/regulatory element combinations. The vector(s) may be inthe form of, for example, a plasmid or a virus, and can be used, aloneor in combination with other vectors, to provide transformed tobaccoplants, using transformation methods as described below to incorporatetransgenes into the genetic material of the tobacco plant(s).

Any transgene(s) known in the art may be introduced into a tobaccoplant, tissue, cell or protoplast according to the present invention,e.g., to improve commercial or agronomic traits, herbicide resistance,disease resistance (e.g., to a bacterial fungal or viral disease),insect resistance, nematode resistance, yield enhancement, nutritionalquality (e.g., oil starch and protein content or quality), leafcharacteristics (color, shape, size, number, angle), and alteredreproductive capability (e.g., male sterility) or chemical composition(e.g., nicotine, total alkaloids, reducing sugars). Alternatively, atransgene may be introduced for the production of recombinant proteins(e.g., enzymes) or metabolites.

In particular embodiments of the invention a transgene conferringherbicide resistance, insect resistance, or disease resistance isintroduced into the tobacco plant. Alternatively, a transgene conferringmale sterility is introduced.

A. Expression Vectors for Tobacco Transformation.

1. Genetic Markers.

Expression vectors typically include at least one genetic marker,operably linked to a regulatory element (a promoter, for example) thatallows transformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker, or by positive selection, i.e., screening for theproduct encoded by the genetic marker. Many commonly used selectablemarker for plant transformation are well known in the transformationart, and include, for example, nucleic acids that code for enzymes thatmetabolically detoxify a selective chemical agent which may be anantibiotic or a herbicide, or nucleic acids that encode an alteredtarget which is insensitive to the inhibitor. A few positive selectionmethods are also known in the art.

One commonly used selectable marker for plant transformation is neomycinphosphotransferase II (npfII), isolated from transposon Tn5, which whenplaced under the control of plant regulatory signals confers resistanceto kanamycin (Fraley et al., (1983) Proc. Natl. Acad. Sci. U.S.A. 80:4803). Another commonly used selectable marker is hygromycinphosphotransferase, which confers resistance to the antibiotichygromycin (Vanden Elzen et al., (1985) Plant Mol. Biol. 5: 299).

Additional selectable markers of bacterial origin that confer resistanceto antibiotics include gentamycin acetyl transferase, streptomycinphosphotransferase, aminoglycoside-3′-adenyl transferase, the bleomycinresistance determinant (Hayford et al., (1988) Plant Physiol. 86: 1216;Jones et al., (1987) Mol. Gen. Genet., 210: 86; Svab et a/., (1990)Plant Mol. Biol. 14: 197; Hille et al., (1986) Plant Mol. Biol. 7: 175).Other selectable markers confer resistance to herbicides such asglyphosate, glufosinate or bromoxynil (Comai et al., (1985) Nature 317:741; Gordon-Kamm et al., (1990) Plant Cell 2: 603; and Stalker et al.,(1988) Science 242: 419).

Selectable markers for plant transformation that are not of bacterialorigin include, for example, mouse dihydrofolate reductase, plant5-eno/pyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase (Eichholtz et al., (1987) Somatic Cell Mol. Genet. 13: 67; Shahet al., (1986) Science 233: 478; Charest et al., (1990) Plant Cell Rep.8: 643).

Another class of markers for plant transformation requires screening ofpresumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These markers are particularly useful to quantify orvisualize the spatial pattern of expression in specific tissues and arefrequently referred to as reporters because they can be fused to anucleic acid or regulatory sequence for the investigation of nucleicacid expression. Commonly used reporters for screening presumptivelytransformed cells include β-glucuronidase (GUS), β-galactosidase,luciferase and chloramphenicol acetyltransferase (Jefferson, R. A.,(1987) Plant Mol. Biol. Rep. 5: 387; Teeri et al., (1989) EMBO J 8: 343;Koncz et al., (1987) Proc. Natl. Acad. Sci. U.S.A. 84:131; De Block etal., (1984) EMBO J. 3: 1681).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are also available (Molecular ProbesPublication 2908, Imagene Green™, p. 1-4 (1993) and Naleway et al.,(1991) J. Cell Biol. 115: 15). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of luciferase as aselectable marker.

In addition, a nucleic acid encoding Green Fluorescent Protein (GFP) hasbeen utilized as a marker for nucleic acid expression in prokaryotic andeukaryotic cells (Chalfie et al., (1994) Science 263: 802). GFP andmutants of GFP may be used as screenable markers.

2. Promoters.

Nucleic acids included in expression vectors are typically driven by anucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation art, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, the term “promoter” refers to a region of a nucleotidesequence that incorporates the necessary signals for the efficientexpression of a coding sequence. This may include sequences to which anRNA polymerase binds, but is not limited to such sequences and caninclude regions to which other regulatory proteins bind together withregions involved in the control of protein translation and can alsoinclude coding sequences. A “plant promoter” is a promoter capable ofinitiating transcription in plant cells. Such promoters include thosethat drive expression of a nucleotide sequence constitutively, thosethat drive expression when induced, and those that drive expression in atissue- or developmentally specific manner, as these various types ofpromoters are known in the art.

(A) Constitutive Promoters.

Thus, for example, in some embodiments of the invention, a constitutivepromoter can be used to drive the expression of a transgene in a plantcell. A constitutive promoter is an unregulated promoter that allows forcontinual transcription of its associated coding sequence. Thus,constitutive promoters are generally active under most environmentalconditions, in most or all cell types and in most or all states; ofdevelopment or cell differentiation.

Any constitutive promoter functional in a plant can be utilized in theinstant invention. Exemplary constitutive promoters include, but are notlimited to, the promoters from plant viruses including, but not limitedto, the 35S promoter from CaMV (Odell et al., Nature 313: 810(1985));figwort mosaic virus (FMV) 35S promoter (P-FMV35S, U.S. Pat. Nos.6,051,753 and 6,018,100); the enhanced CaMV35S promoter (e35S); the 1′-or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens; thenopaline synthase (NOS) and/or octopine synthase (OCS) promoters, whichare carried on tumor-inducing plasmids of Agrobacterium tumefaciens(Ebert et al., Proc. Natl. Acad. Sci. (U.S.A.), 84:5745 5749, 1987);actin promoters including, but not limited to, rice actin (McElroy etal., Plant Cell 2: 163 (1990); U.S. Pat. No. 5,641,876); histonepromoters; tubulin promoters; ubiquitin and polyubiquitin promoters((Sun and Callis, Plant J., 11(5):1017-1027 (1997)); Christensen et al.,Plant Mol. Biol 12: 619(1989) and Christensen et al., Plant Mol. Biol.18: 675(1992)); pEMU (Last et al., Theor. Appl. Genet. 81: 581(1991));the mannopine synthase promoter (MAS) (Velten et al., EMBO J. 3:2723(1984)); maize H3 histone (Lepelit et al., Mol. Gen. Genet. 231: 276(1992) and Atanassova et al., Plant Journal 2: 291 (1992)); the ALSpromoter, a XbaI/NcoI fragment 5′ to the Brassica napus ALS3 structuralgene (or a nucleotide sequence that has substantial sequence similarityto said XbaI/NcoI fragment); ACT11 from Arabidopsis (Huang et al., PlantMol. Biol. 33:125-139 (1996)); Cat3 from Arabidopsis (GenBank No.U43147, Zhong et al., Mol. Gen. Genet. 251:196-203 (1996)); GPc1 frommaize (GenBank No. X15596, Martinez et al., J. Mol. Biol. 208:551-565(1989)); and Gpc2 from maize (GenBank No. U45855, Manjunath et al.,Plant Mol. Biol. 33:97-112 (1997)).

(B) Inducible Promoters.

In some embodiments of the present invention, an inducible promoter canbe used to drive the expression of a transgene. Inducible promotersactivate or initiate expression only after exposure to, or contact with,an inducing agent. Inducing agents include, but are not limited to,various environmental conditions (e.g., pH, temperature), proteins andchemicals. Examples of environmental conditions that can affecttranscription by inducible promoters include pathogen attack, anaerobicconditions, extreme temperature and/or the presence of light. Examplesof chemical inducing agents include, but are not limited to, herbicides,antibiotics, ethanol, plant hormones and steroids. Any induciblepromoter that is functional in a plant can be used in the instantinvention (see, Ward et al., (1993) Plant Mol. Biol. 22: 361 (1993));Exemplary inducible promoters include, but are not limited to, that fromthe ACEI system, which responds to copper (Melt et al., PNAS 90: 4567(1993)); the In2 nucleic acid from maize, which responds tobenzenesulfonamide herbicide safeners (Hershey et al., (1991) Mol. Gen.Genetics 227: 229 (1991) and Gatz et al., Mol. Gen. Genetics 243: 32(1994)); a heat shock promoter, including, but not limited to, thesoybean heat shock promoters Gmhsp 17.5-E, Gmhsp 17.2-E and Gmhsp 17.6-Land those described in U.S. Pat. No. 5,447,858; the Tet repressor fromTn10 (Gatz et al., Mol. Gen. Genet. 227: 229 (1991)) and thelight-inducible promoter from the small subunit of ribulose bisphosphatecarboxylase (ssRUBISCO). Other examples of inducible promoters include,but are not limited to, those described by Moore et al. (Plant J.45:651-683 (2006)). Additionally, some inducible promoters respond to aninducing agent to which plants do not normally respond. An example ofsuch an inducible promoter is the inducible promoter from a steroidhormone gene, the transcriptional activity of which is induced by aglucocorticosteroid hormone (Schena et al., Proc. Natl. Acad. Sci.U.S.A. 88: 421 (1991)).

(C) Tissue-Specific or Tissue-Preferred Promoters.

In further embodiments of the present invention, a tissue-specificpromoter can be used to drive the expression of a transgene in aparticular tissue in the transgenic plant. Tissue-specific promotersdrive expression of a nucleic acid only in certain tissues or celltypes, e.g., in the case of plants, in the leaves, stems, flowers andtheir various parts, roots, fruits and/or seeds, etc. Thus, plantstransformed with a nucleic acid of interest operably linked to atissue-specific promoter produce the product encoded by the transgeneexclusively, or preferentially, in a specific tissue or cell type.

Any plant tissue-specific promoter can be utilized in the instantinvention. Exemplary tissue-specific promoters include, but are notlimited to, a root-specific promoter, such as that from the phaseolingene (Murai et al., (1983) Science 23: 476 and Sengupta-Gopalan et al.,(1985) Proc. Natl. Acad. Sci. USA 82: 3320); a leaf-specific andlight-induced promoter such as that from cab or rubisco (Simpson et al.(1985) EMBO J. 4: 2723 and Timko et al., (1985) Nature 318: 579); thefruit-specific E8 promoter from tomato (Lincoln et al. Proc. Nat'l.Acad. Sci. USA 84: 2793-2797 (1988); Deikman et al. EMBO J. 7: 3315-3320(1988); Deikman et al. Plant Physiol. 100: 2013-2017 (1992);seed-specific promoters of, for example, Arabidopsis thaliana (Krebberset al. (1988) Plant Physiol. 87:859); an anther-specific promoter suchas that from LAT52 (Twell et al. (1989) Mol. Gen. Genet. 217: 240) orEuropean Patent Application No 344029, and those described by Xu et al.(Plant Cell Rep. 25:231-240 (2006)) and Gomez et al. (Planta 219:967-981(2004)); a pollen-specific promoter such as that from Zm13 (Guerrero etal., (1993) Mol. Gen. Genet. 224: 161), and those described by Yamaji etal. (Plant Cell Rep. 25:749-57 (2006)) and Okada et al. (Plant CellPhysiol. 46:749-802 (2005)); a pith-specific promoter, such as thepromoter isolated from a plant TrpA gene as described in InternationalPCT Publication No. WO93/07278; and a microspore-specific promoter suchas that from apg (Twell et al. (1993) Sex. Plant Reprod. 6: 217).Exemplary green tissue-specific promoters include the maize phosphoenolpyruvate carboxylase (PEPC) promoter, small subunit ribulosebis-carboxylase promoters (ssRUBISCO) and the chlorophyll a/b bindingprotein promoters.

3. Signal Sequences for Targeting Proteins to Subcellular Compartments.

Transport of proteins produced by transgenes to a subcellularcompartment such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall or mitochondrion, or for secretion into the apoplast, may beaccomplished by means of operably linking a nucleotide sequence encodinga signal sequence typically at the 5′ and/or 3′ region of a sequenceencoding the protein of interest. Association of targeting sequenceswith the coding sequence may determine, during protein synthesis andprocessing, where the encoded protein is ultimately compartmentalized.The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art (see, forexample, Becker et al., (1992) Plant Mol. Biol. 20: 49; Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al., (1987)Plant Mol. Biol. 9: 3; Lerner et al., (1989) Plant Physiol. 91: 124;Fontes et al., (1991) Plant Cell 3: 483; Matsuoka et al., (1991) Proc.Natl. Acad. Sci. 88: 834; Gould et al., (1989) J. Cell Biol 108: 1657;Creissen et al., (1991) Plant J. 2: 129; Kalderon et al., (1984) Cell39: 499; Stiefel et al., (1990) Plant Cell 2: 785).

B. Foreign Nucleic Acids that May be Introduced into Tobacco Plants.

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants, which areharvested in a conventional manner, and a foreign protein can then beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, (1991) Anal. Biochem. 114:92.

According to embodiments of the invention, a transgenic tobacco plant isprovided for commercial production of foreign protein. A genetic map canbe generated, for example, via conventional Restriction Fragment LengthPolymorphisms (RFLP), Polymerase Chain Reaction (PCR) analysis, andSimple Sequence Repeats (SSR), which identifies the approximatechromosomal location of the integrated DNA molecule. For exemplarymethodologies in this regard, see Glick and Thompson, METHODS IN PLANTMOLECULAR BIOLOGY AND BIOTECHNOLOGY 269-284 (CRC Press, Boca Raton,1993). Map information concerning chromosomal location is useful forproprietary protection of a subject transgenic plant. If unauthorizedpropagation is undertaken and crosses made with other germplasm, the mapof the integration region can be compared to similar maps for suspectplants, to determine if the latter have a common parentage with thesubject plant. Map comparisons would involve hybridizations, RFLP, PCR,SSR and sequencing, all of which are conventional techniques.

Likewise, by means of the present invention, nucleic acids of agronomicimportance can be expressed in transformed plants. More particularly,plants can be genetically engineered to express various phenotypes ofagronomic interest. Exemplary nucleic acids implicated in this regardinclude, but are not limited to, those described below.

As an example, a nucleic acid conferring male sterility may betransformed into cultivar AOB 175. There are several methods ofconferring genetic male sterility available, such as multiple mutantnucleic acids at separate locations within the genome that confer malesterility, as disclosed in U.S. Pat. Nos. 4,654,465 and 4,727,219 toBrar et al. and chromosomal translocations as described by Patterson inU.S. Pat. Nos. 3,861,709 and 3,710,511. Examples include: (A)Introduction of a deacetylase nucleic acid under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT (WO 01/29237). (B) Introduction of various stamen-specificpromoters (WO 92/13956, WO 92/13957). (C) Introduction of the barnaseand the barstar nucleic acids (Paul et al. Plant Mol. Biol. 19:611 622,1992). For additional examples of nuclear male and female sterilitysystems and nucleic acids, see also, Nikova et al., Plant Cell, Tissueand Organ Culture 27:289-295 (1991); Nikova et al., Euphytica 94:375-378(1997); Atanassov et al., Theoretical and Applied Genetics 97:982-985(1998); Berbec, A. Bull. Spec. Coresta, Lisbon Congress, p. 79, abstractAP30, (2000); U.S. Pat. No. 5,859,341; U.S. Pat. No. 6,297,426; U.S.Pat. No. 5,478,369; U.S. Pat. No. 5,824,524; U.S. Pat. No. 5,850,014;and U.S. Pat. No. 6,265,640; all of which are hereby incorporated byreference.

In an additional embodiment, a transgene whose expression results orcontributes to a desired trait to be transferred to cultivar AOB 175comprises a nucleic acid encoding an insecticidal protein, such as, forexample, a crystal protein of Bacillus thuringiensis or a vegetativeinsecticidal protein from Bacillus cereus, such as VIP3 (see, forexample, Estruch et al. (1997) Nat Biotechnol 15:137).

In a further embodiment, a transgene introduced into cultivar AOB 175comprises a nucleic acid conferring herbicide tolerance whose expressionrenders plants of cultivar AOB 175 tolerant to the herbicide. Forexample, expression of an altered acetohydroxyacid synthase (AHAS)enzyme confers upon plants tolerance to various imidazolinone orsulfonamide herbicides (U.S. Pat. No. 4,761,373). In a still furtherembodiment, a nucleic acid conferring tolerance to imidazolinones orsulfonylurea herbicides is transferred into cultivar AOB 175. Expressionof a mutant acetolactate synthase (ALS) will render the plants resistantto inhibition by sulfonylurea herbicides (U.S. Pat. No. 5,013,659).

U.S. Pat. No. 4,975,374 describes plant cells and plants containing anucleic acid encoding a mutant glutamine synthetase (GS) which confersresistance to herbicides that are known to inhibit GS, e.g.,phosphinothricin and methionine sulfoximine. In addition, expression ofa Streptomyces bar nucleic acid encoding a phosphinothricin acetyltransferase results in tolerance to the herbicide phosphinothricin orglufosinate (U.S. Pat. No. 5,489,520). U.S. Pat. No. 5,162,602 disclosesplants tolerant to inhibition by cyclohexanedione andaryloxyphenoxypropanoic acid herbicides. The tolerance is conferred byan altered acetyl coenzyme A carboxylase (ACCase). U.S. Pat. No.5,554,798 discloses transgenic glyphosate tolerant plants, whichtolerance is conferred by an altered 5-enolpyruvyl-3-phosphoshikimate(EPSP) synthase nucleic acid. In another particular embodiment,tolerance to a protoporphyrinogen oxidase inhibitor is achieved byexpression of a tolerant protoporphyrinogen oxidase enzyme in plants(U.S. Pat. No. 5,767,373). In another particular embodiment, a nucleicacid transferred into cultivar AOB 175 comprises a transgene conferringtolerance to a herbicide and at least one other transgene conferringanother trait, such as for example, insect resistance or tolerance toanother herbicide.

Other illustrative transgenes are set forth below.

1. Transgenes that Confer Resistance to Pests or Disease and thatEncode:

(A) Plant disease resistance. Plant defenses are often activated byspecific interaction between the product of a nucleic acid coding fordisease resistance gene (R) in the plant and the product of acorresponding nucleic acid coding for avirulence (Avr) in the pathogen.A plant variety can be transformed with a cloned nucleic acid conferringresistance in order to engineer plants that are resistant to specificpathogens (see, for example, Jones et al., (1994) Science 266: 789,cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum;Martin et al., (1993) Science 262: 1432, tomato Pto gene for resistanceto Pseudomonas syringae pv.; Mindrinos et al., (1994) Cell 78: 1089,Arabidopsis RSP2 nucleic acid encoding resistance to Pseudomonassyringae).

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon (see, for example, Geiser et al.,(1986) Gene 48: 109, disclosing the cloning and nucleotide sequence ofBt δ-endotoxin). Moreover, DNA molecules encoding δ-endotoxin can bepurchased from American Type Culture Collection (Rockville, Md.), forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998. Otherexamples of Bacillus thuringiensis transgenes being geneticallyengineered are given in the following patents and patent applicationsand hereby are incorporated by reference for this purpose: U.S. Pat.Nos. 5,188,960; 5,689,052; 5,880,275; WO 91/14778; WO 99/31248; WO01/12731; WO 99/24581; WO 97/40162 and U.S. application Ser. Nos.10/032,717; 10/414,637; and 10/606,320.

(C) A lectin (see, for example, the disclosure by Van Damme et al.,(1994) Plant Molec. Biol. 24: 25), which discloses the nucleotidesequences of several Clivia miniata mannose-binding lectins.

(D) A vitamin-binding protein such as avidin (see PCT publication WO93/06487). This publication teaches the use of avidin and avidinhomologues as larvicides against insect pests.

(E) An enzyme inhibitor, for example, a protease inhibitor or an amylaseinhibitor (see, for example, Abe et al., (1987) J. Biol. Chem. 262:16793, nucleotide sequence of rice cysteine proteinase inhibitor; Huubet al., (1993) Plant Molec. Biol. 21: 985; nucleotide sequence of cDNAencoding tobacco proteinase inhibitor 1; and Sumitani et al., (1993)Biosci. Biotech. Biochem. 57: 1243, nucleotide sequence of Streptomycesnitrosporeus amylase inhibitor).

(F) An insect-specific hormone or pheromone such as an ecdysteroid orjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof (see, for example, the disclosure ofHammock et al., (1990) Nature 344: 458, of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone).

(G) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest (for example, see thedisclosures of Regan, (1994) J. Biol. Chem. 269: 9, expression cloningyields DNA coding for insect diuretic hormone receptor; Pratt et al.,(1989) Biochem. Biophys. Res. Comm. 163: 1243, an allostatin isidentified in Diploptera puntata). Chattopadhyay et al. (2004) Crit.Rev. Microbiol. 30 (1): 33 54 2004; Zjawiony (2004) J. Nat. Prod. 67(2): 300 310; Carlini & Grossi-de-Sa (2002) Toxicon, 40 (11): 1515 1539;Ussuf et al. (2001) Curr. Sci. 80 (7): 847 853; and Vasconcelos &Oliveira (2004) Toxicon 44 (4): 385 403 See also U.S. Pat. No. 5,266,317to Tomalski et al., which discloses nucleic acis encodinginsect-specific, paralytic-neurotoxins.

(H) An insect-specific venom produced in nature by a snake, a wasp, orthe like (see, e.g., Pang et al., (1992) Gene 116: 165, for disclosureof heterologous expression in plants of a nucleic acid encoding ascorpion insectotoxic peptide).

(I) An enzyme responsible for an hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(J) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic (see PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase). DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152 (see also Kramer et al., (1993)Insect Biochem. Molec. Biol. 23: 691, which describes the nucleotidesequence of a cDNA encoding tobacco hookworm chitinase, and Kawalleck etal., (1993) Plant Molec. Biol. 21: 673, which provides the nucleotidesequence of parsley ubi4-2 polyubiquitin).

(K) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., (1994) Plant Molec. Biol. 24: 757, ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., (1994) Plant Physio. 104: 1467, which provides the nucleotidesequence of a maize calmodulin cDNA clone.

(L) A hydrophobic moment peptide (see PCT application WO 95/16776 whichdiscloses peptide derivatives of Tachyplesin which inhibit fungal plantpathogens, and PCT application WO 95/18855 which teaches syntheticantimicrobial peptides that confer disease resistance).

(M) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., (1993) Plant Sci. 89: 43),of heterologous expression of a cecropin-β lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(N) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the nucleic acid encoding the coatprotein is derived, as well as by related viruses (see Beachy et al.,(1990) Ann. Rev. Phytopathol. 28: 451). Coat protein-mediated resistancehas been conferred upon transformed plants against alfalfa mosaic virus,cucumber mosaic virus, tobacco streak virus, potato virus X, potatovirus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaicvirus (Id.).

(O) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect (Cf.Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994); enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(P) A virus-specific antibody (see, for example, Taviadoraki et al.,(1993) Nature 366: 469; showing that transgenic plants expressingrecombinant antibody are protected from virus attack).

(Q) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase (see Lamb et al., (1992)Bio/Technology 10: 1436). The cloning and characterization of a nucleicacid which encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., (1992) Plant J. 2: 367.

(R) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., (1992) Bio/Technology 10: 305, have shown thattransgenic plants expressing the barley ribosome-inactivating nucleicacid have an increased resistance to fungal disease.

(S) Nucleic acids involved in the Systemic Acquired Resistance (SAR)Response and/or the pathogenesis related nucleic acids. Briggs, S.,Current Biology, 5(2) (1995), Pieterse & Van Loon (2004) Curr. Opin.Plant Bio. 7(4):456 64 and Somssich (2003) Cell 113(7):815 6.

(T) Nucleic acids encoding resistance to fungi (Cornelissen andMelchers, Pl. Physiol. 101:709 712, (1993) and Parijs et al., Planta183:258 264, (1991) and Bushnell et al., Can. J. Plant Pathol. 20(2):137149 (1998). Also see U.S. application Ser. No. 09/950,933.

2. Transgenes that Confer Resistance to a Herbicide, for Example:

(A) An herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary transgenes or nucleic acidsin this category code for mutant ALS or AHAS enzyme as described, forexample, by Lee et al., (1988) EMBO J. 7: 1241, and Miki et al., (1990)Theor. Appl. Genet. 80: 449, respectively.

(B) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA nucleic acids) andother phosphono compounds such as glufosinate (phosphinothricin acetyltransferase (PAT) and Streptomyces hygroscopicus phosphinothricin acetyltransferase (bar) nucleic acids), and pyridinoxy or phenoxy proprionicacids and cycloshexones (ACCase inhibitor-encoding nucleic acids). See,for example, U.S. Pat. No. 4,940,835 to Shah et al., which discloses thenucleotide sequence of a form of EPSP which can confer glyphosateresistance. A DNA molecule encoding a mutant aroA can be obtained underATCC accession No. 39256, and the mutant nucleotide sequence isdisclosed in U.S. Pat. No. 4,769,061 to Comai. European patentapplication No. 0 333 033 to Kumada et al. and U.S. Pat. No. 4,975,374to Goodman et al. discloses nucleotide sequences encoding glutaminesynthetase which confers resistance to herbicides such asL-phosphinothricin. The nucleotide sequence encoding aphosphinothricin-acetyl-transferase is provided in European applicationNo. 0 242 246 to Leemans et al. De Greef et al., (1989) Bio/Technology7: 61, describes the production of transgenic plants that expresschimeric bar coding for phosphinothricin acetyl transferase activity.Exemplary nucleic acids conferring resistance to phenoxy proprionicacids and cycloshexones, such as sethoxydim and haloxyfop, are theAcc1-S1, Acc1-S2 and Acc1-S3 nucleic acids described by Marshall et al.,(1992) Theor. Appl. Genet. 83: 435.

(C) An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+) and a benzonitrile (nitrilase). Przibilla et al., (1991) PlantCell 3: 169, describe the transformation of Chlamydomonas with plasmidsencoding mutant psbA. Nucleic acids encoding nitrilase are disclosed inU.S. Pat. No. 4,810,648 to Stalker, and these nucleic acids areavailable under ATCC Accession Nos. 53435, 67441 and 67442. Cloning andexpression of DNA coding for a glutathione S-transferase is described byHayes et al., (1992) Biochem. J. 285: 173.

3. Transgenes that Confer or Contribute to a Value-Added Trait, such as:

(A) Decreased phytate content: Introduction of a phytase-encodingnucleic acid would enhance breakdown of phytate, adding more freephosphate to the transformed plant. For example, see Van Hartingsveldtet al., (1993) Gene 127: 87, for a disclosure of the nucleotide sequenceof an Aspergillus niger phytase.

(B) Modified carbohydrate composition effected, for example, bytransforming plants with a nucleic acid encoding an enzyme that altersthe branching pattern of starch (see Shiroza et al., (1998) J.Bacteriol. 170: 810, nucleotide sequence of Streptococcus mutansfructosyltransferase; Steinmetz et al., (1985) Mol. Gen. Genet. 200:220, nucleotide sequence of Bacillus subtilis levansucrase; Pen et al.,(1992) Bio/Technology 10: 292, production of transgenic plants thatexpress Bacillus licheniformis α-amylase; Elliot et al., (1993) PlantMolec. Biol. 21: 515, nucleotide sequences of tomato invertase; Søgaardet al., (1993) J. Biol. Chem. 268: 22480, site-directed mutagenesis ofbarley α-amylase nucleic acid; and Fisher et al., (1993) Plant Physiol.102: 1045, maize endosperm starch branching enzyme II).

Those skilled in the art will appreciate that the transgenes describedabove may also be transferred into tobacco plants using conventionalbreeding techniques as known in the art and as described herein.

As a further alternative, the transgene can encode an antisense RNAmolecule or any other non-translated RNA as known in the art. In afurther alternative embodiment, the transgene effects gene suppressionin the host plant.

C. Methods for Tobacco Transformation.

Plants can be transformed according to the present invention using anysuitable method known in the art. Intact plants, plant tissue, explants,meristematic tissue, protoplasts, callus tissue, cultured cells, and thelike may be used for transformation depending on the plant species andthe method employed. Procedures for transforming a wide variety of plantspecies are well known and routine in the art and described throughoutthe literature. Such methods include, but are not limited to,transformation via bacterial-mediated nucleic acid delivery,viral-mediated nucleic acid delivery, silicon carbide or nucleic acidwhisker-mediated nucleic acid delivery, liposome mediated nucleic aciddelivery, microinjection, microparticle bombardment, electroporation,sonication, infiltration, PEG-mediated nucleic acid uptake, as well asany other electrical, chemical, physical (mechanical) and/or biologicalmechanism that results in the introduction of nucleic acid into theplant cell, including any combination thereof. General guides to variousplant transformation methods known in the art include Miki et al.(“Procedures for Introducing Foreign DNA into Plants” in Methods inPlant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) andRakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

Bacterial mediated nucleic acid delivery includes but is not limited toDNA delivery by Agrobacterium spp. and is described, for example, inHorsch et al. (Science 227:1229 (1985); Ishida et al. (NatureBiotechnol. 14:745 750 (1996); and Fraley et al. (Proc. Natl. Acad. Sci.80: 4803 (1983)). Transformation by various other bacterial species isdescribed, for example, in Broothaerts et al. (Nature 433:629-633(2005)).

Physical delivery of nucleotide sequences via microparticle bombardmentis also well known and is described, for example, in Sanford et al.(Methods in Enzymology 217:483-509 (1993)) and McCabe et al. (Plant CellTiss. Org. Cult. 33:227-236 (1993)).

Another method for physical delivery of nucleic acid to plants issonication of target cells. This method is described, for example, inZhang et al. (Bio/Technology 9:996 (1991)). Nanoparticle-mediatedtransformation is another method for delivery of nucleic acids intoplant cells (Radu et al., J. Am. Chem. Soc. 126: 13216-13217 (2004);Torney, et al. Society for In Vitro Biology, Minneapolis, Minn. (2006)).Alternatively, liposome or spheroplast fusion can be used to introducenucleotide sequences into plants. Examples of the use of liposome orspheroplast fusion are provided, for example, in Deshayes et al. (EMBOJ., 4:2731 (1985), and Christou et al. (Proc Natl. Acad. Sci. U.S.A.84:3962 (1987)). Direct uptake of nucleic acid into protoplasts usingCaCl₂ precipitation, polyvinyl alcohol or poly-L-ornithine is described,for example, in Hain et al. (Mol. Gen. Genet. 199:161 (1985)) and Draperet al. (Plant Cell Physiol. 23:451 (1982)). Electroporation ofprotoplasts and whole cells and tissues is described, for example, inDonn et al. (In Abstracts of VIIth International Congress on Plant Celland Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin et al. (PlantCell 4:1495-1505 (1992)); Spencer et al. (Plant Mol. Biol. 24:51-61(1994)) and Fromm et al. (Proc. Natl. Acad. Sci. 82: 5824 (1985)).Polyethylene glycol (PEG) precipitation is described, for example, inPaszkowski et al. (EMBO J. 3:2717 2722 (1984)). Microinjection of plantcell protoplasts or embryogenic callus is described, for example, inCrossway (Mol. Gen. Genetics 202:179-185 (1985)). Silicon carbidewhisker methodology is described, for example, in Dunwell et al.(Methods Mol. Biol. 111:375-382 (1999)); Frame et al. (Plant J.6:941-948 (1994)); and Kaeppler et al. (Plant Cell Rep. 9:415-418(1990)).

Plant cells, which have been transformed by any method known in the art,can also be regenerated to produce intact plants using known techniques.

Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMilan Publishing Co.New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. II,1986). It is known that practically all plants can be regenerated fromcultured cells or tissues.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts or a petri platecontaining transformed explants is first provided. Callus tissue isformed and shoots may be induced from callus and subsequently root.Alternatively, somatic embryo formation can be induced in the callustissue. These somatic embryos germinate as natural embryos to formplants. The culture media will generally contain various amino acids andplant hormones, such as auxin and cytokinins. A large number of plantshave been shown capable of regeneration from transformed individualcells to obtain transgenic whole plants.

The regenerated plants are transferred to standard soil conditions andcultivated in a conventional manner. The plants are grown and harvestedusing conventional procedures.

The foregoing methods for transformation may be used for producingtransgenic inbred lines. Transgenic inbred lines can then be crossed,with another (non-transformed or transformed) inbred line, in order toproduce a transgenic hybrid tobacco plant. Alternatively, a genetictrait that has been engineered into a particular tobacco line using theforegoing transformation techniques can be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach can be used to movean engineered trait from a non-elite line into an elite tobacco line, orfrom a hybrid tobacco plant containing a foreign nucleic acid in itsgenome into a line or lines, which do not contain that nucleic acid. Asused above, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

VI. PRODUCTS

Tobacco plants, or parts thereof, of the present invention may beutilized in any product containing tobacco including without limitationpipe, cigar and cigarette tobacco, and chewing tobacco, snuff, andtobacco-containing gum and lozenges; and may be in any form includingleaf tobacco, shredded tobacco, cut tobacco, or tobacco extract.Accordingly, some embodiments of the invention provide tobacco productsproduced from the plants of the present invention, or parts thereof. Thetobacco plants of the invention, or parts thereof, can be also used inblends with tobacco from other tobacco varieties to make a tobaccoproduct

VII. INDUSTRIAL APPLICABILITY

This invention is also directed to methods for producing a tobacco plantby crossing a first parent tobacco plant with a second parent tobaccoplant wherein either the first or second parent tobacco plant is atobacco plant of cultivar AOB 175 or a tobacco plant of cultivar AOB 175further comprising one or more additional traits (e.g., single genetraits). Further, both first and second parent tobacco plants can comefrom cultivar AOB 175 or a tobacco plant of cultivar AOB 175 furthercomprising one or more traits (e.g., single gene traits). Thus, any suchmethods using the tobacco cultivar AOB 175 or a tobacco plant of AOB 175further comprising one or more additional traits (e.g., one or moresingle gene traits) are part of this invention: selfing, backcrosses,doubled-haploid production, hybrid production, crosses to populations,and the like. All plants produced using tobacco cultivar AOB 175 ormodified cultivar AOB 175 further comprising one or more additionaltraits (e.g., one or more single gene traits) as a parent are within thescope of this invention. Advantageously, tobacco cultivar AOB 175 ormodified cultivar AOB 175 further comprising one or more additionaltraits (e.g., one or more single gene traits) are used in crosses withother, different, tobacco inbreds to produce first generation (F₁)tobacco hybrid seeds and plants with superior characteristics.

VIII. DEPOSITS

A deposit of at least 2500 seeds of tobacco cultivar AOB 175 has beenmade with the American Type Culture Collection (ATCC), Manassas, Va.20110 USA on Nov. 6, 2008. The deposit has been assigned ATCC AccessionNumber PTA-9589. This deposit of the tobacco cultivar AOB 175 will bemaintained in the ATCC depository, which is a public depository, for aperiod of 30 years, or 5 years after the most recent request, or for theeffective life of the patent, whichever is longer, and will be replacedif it becomes nonviable during that period. Applicants do not waive anyinfringement of their rights granted under this patent or under thePlant Variety Protection Act (7 U.S.C. 2321 et seq.).

Having now described the invention, the same will be illustrated withreference to certain examples, which are included herein forillustration purposes only, and which are not intended to be limiting ofthe invention.

1. A tobacco seed designated AOB 175, representative seed of saidtobacco cultivar AOB 175 having been deposited under ATCC Accession No.PTA-9589.
 2. A tobacco plant, or a part thereof, produced by the seed ofclaim
 1. 3. Pollen of the plant of claim
 2. 4. An ovule of the plant ofclaim
 2. 5. A tobacco plant, or a part thereof, having all thephysiological and morphological characteristics of tobacco cultivar AOB175, the tobacco cultivar AOB 175 having been deposited under ATCCAccession No. PTA-9589.
 6. The tobacco plant of claim 2, wherein saidplant further comprises a nucleic acid conferring male sterility.
 7. Thetobacco plant of claim 5, wherein said plant further comprises a nucleicacid conferring male sterility.
 8. A tissue culture of regenerable cellsof the plant, or part thereof, of claim
 2. 9. The tissue cultureaccording to claim 8, wherein the regenerable cells are from plant partsselected from the group consisting of leaves, pollen, embryos,cotyledons, hypocotyls, roots, root tips, anthers, flowers and a partthereof, ovules, shoots, stems, stalks, pith and capsules or wherein theregenerable cells are callus or protoplasts derived therefrom.
 10. Atobacco plant regenerated from the tissue culture of claim 8 expressingall the morphological and physiological characteristics of tobaccocultivar AOB 175, the tobacco cultivar AOB 175 having been depositedunder ATCC Accession No. PTA-9589.
 11. The tobacco plant of claim 10,wherein said plant further comprises a nucleic acid conferring malesterility.
 12. A method for producing a first generation (F₁) hybridtobacco seed comprising crossing the plant of claim 2 with a differenttobacco plant and harvesting the resultant first generation (F₁) hybridtobacco seed.
 13. An F₁ hybrid tobacco seed produced by the method ofclaim
 12. 14. An F₁ hybrid plant, or apart thereof, grown from the seedof claim
 13. 15. The tobacco plant of claim 14, wherein said plantfurther comprises a nucleic acid conferring male sterility.
 16. A methodfor producing hybrid tobacco seed comprising crossing two tobacco plantsand harvesting the resultant hybrid tobacco seed, wherein at least onetobacco plant is the tobacco plant of claim
 2. 17. A method forproducing an AOB 175-derived tobacco plant comprising: (a) crossingtobacco cultivar AOB 175, representative seed of said tobacco cultivarAOB 175 having been deposited under ATCC Accession No. PTA-9589, with asecond tobacco plant to yield progeny tobacco seed; (b) growing saidprogeny tobacco seed, under plant growth conditions, to yield said AOB175-derived tobacco plant.
 18. An AOB 175-derived tobacco plant, or apart thereof, produced by the method of claim
 17. 19. The tobacco plantof claim 18, wherein said plant further comprises a nucleic acidconferring male sterility.
 20. The tobacco plant, or a part thereof, ofclaim 2 wherein the plant or a part thereof has been transformed so thatits genetic material comprises one or more transgenes operably linked toone or more regulatory elements.
 21. A method for producing a tobaccoplant that contains in its genetic material one or more transgenes,comprising crossing the tobacco plant of claim 20 with either a secondplant of another tobacco line, or a non-transformed tobacco plant ofclaim 2, wherein progeny are produced, so that the genetic material ofthe progeny that result from the cross comprises the transgene(s)operably linked to one or more regulatory elements.
 22. The method ofclaim 21, wherein the transgene is selected from the group consisting oftransgenes the expression of which confers herbicide resistance, insectresistance, disease resistance and/or male sterility.
 23. A tobaccoplant, or a part thereof, produced by the method of claim
 22. 24. Thetobacco plant, or a part thereof, of claim 8 wherein the plant or a partthereof has been transformed so that its genetic material comprises oneor more transgenes operably linked to one or more regulatory elements.25. A method for producing a tobacco plant that contains in its geneticmaterial one or more transgenes, comprising crossing the tobacco plantof claim 24 with either a second plant of another tobacco line, or anon-transformed tobacco plant of claim 8, so that the genetic materialof the progeny that result from the cross comprises the transgene(s)operably linked to one or more regulatory elements.
 26. The method ofclaim 25, wherein the transgene is selected from the group consisting ofa transgene the expression of which confers herbicide resistance, insectresistance, disease resistance and/or male sterility.
 27. A tobaccoplant, or a part thereof, produced by the method of claim
 26. 28. Amethod for developing a tobacco plant in a tobacco plant breedingprogram using plant breeding techniques, which include employing atobacco plant, or a part thereof, as a source of plant breedingmaterial, comprising: using the tobacco plant, or a part thereof ofclaim 2 as a source of said breeding material.
 29. The method fordeveloping a tobacco plant breeding program of claim 28, wherein saidplant breeding techniques are selected from the group consisting ofrecurrent selection, backcrossing, pedigree breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection, double haploid breeding, single seed descent, multiple seeddescent, and transformation.
 30. A tobacco plant, or a part thereof,produced by the method of claim
 28. 31. A tobacco product produced fromthe tobacco plant of claim
 2. 32. A tobacco product produced from thetobacco plant of claim
 5. 33. A tobacco product produced from thetobacco plant of claim 14.